In the last decade, the genomes of over 50 organisms have been sequenced, resulting in a vast increase in the number of known proteins. Characterization of the abundance, post-translational modification, structure, and function of these proteins represents a major challenge for genomic analysis. Understanding protein conformation, interactions and ligand binding is essential to all biological inquiry. Defining protein structure in molecular detail constitutes a particularly difficult task. Accurate measurement of these properties currently requires high-resolution physical methods. While X-ray crystallographic and nuclear magnetic resonance techniques are extremely powerful, they require large investments of time and material, and are limited in their application to large protein complexes, membrane proteins and other insoluble or partially folded polypeptides. Many proteins and protein complexes prove unsuitable for NMR and X-ray work. In order to rapidly obtain functional information for a large number of sequences, a general and efficient tool for probing protein conformation is required.
In principle, protein footprinting is an option for studying protein structure, but it has been far less successful than the corresponding techniques developed for nucleic acids (Galas D. J., A. Schmitz, Nucleic Acids Res. 5, 3157 (1978)). Conventional protein footprinting involves the treatment of a protein of interest with an enzymatic protease which cleaves the protein backbone at accessible positions. The protein fragments generated under various conditions are analyzed (e.g. in the presence or absence of substrate or ligand) to determine which regions of the protein have changed their susceptibility to the protease. Because of the chemical heterogeneity of the amino-acid side chains, no reagent (chemical or enzyme) exists with the ability to cleave the protein backbone uniformly under native conditions. Furthermore, protein separation techniques such as SDS-PAGE do not provide the single-monomer resolution of the urea-acrylamide gels used for the separation of nucleic acids, thus complicating the analysis of observed cleavage patterns. Finally, because of the cooperative nature of protein unfolding, proteolytic cleavage at one site often leads to a global loss of structure and to increased cleavage at other sites in the same molecule, resulting in artifactual data.
Footprinting by chemical modification of amino acid side chains represents a different approach to the problem. Modification of side chains is carried out under native conditions, while detection of modifications can be performed under arbitrary conditions. The susceptibility of each side chain to modification reports its solvent accessibility. Acylation of lysine residues (Doonan S., H. M. Fahmy, Eur. J. Biochem. 56,421, 1975); Hanai, R., J. C. Wang, Proc. Natl. Acad. Sci. U.S.A. 91, 11904, 1994), oxidation of methionine residues (de Arruda, M. V. et. al., J. Biol. Chem. 267, 13079, 1992), and alkylation of cysteine residues (Doering, D. S., P. Matsudaira, Biochemistry 35, 12677, 1996; Tu, B. P., J. C. Wang, Proc. Natl. Acad. Sci. U.S.A. 96, 4862, 1999) have been used previously to footprint protein structures. In general, these studies have been limited in scope, however, as they examine only a few naturally-occurring residues, or require extensive site-directed mutagenesis to introduce additional structural probes.
Thus, there is a need to develop a method for protein analysis which allows for the rapid structural characterization of proteins and protein complexes at a large number of sites distributed throughout the protein.
Literature of Interest
Ha and Loh (Nat. Struct. Biol., 1998. 5:730-7) and Young, et al. (Proc Natl Acad Sci USA, 2000 97: p. 5802-6) may also be of interest.